Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

LC-MS/(MS) confirmatory doping control analysis of intact phase II metabolites of methenolone and mesterolone after Girard's Reagent T derivatization.

In the present study, the application and evaluation of Girard's Reagent T (GRT) derivatization for the simultaneous detection and significantly important identification of different phase II methenolone and mesterolone metabolites by LC-MS/(MS) are presented. For the LC-MS analysis of target analytes two complementary isolation methods were developed; a derivatization and shoot method in which native urine is diluted with derivatization reagent and is injected directly to LC-MS and a liquid-liquid extraction method, using ethyl acetate at pH 4.5, for the effective isolation of both sulfate and glucuronide metabolites of the named steroids as well as of their free counterparts. For the evaluation of the proposed protocols, urine samples from methenolone and mesterolone excretion studies were analyzed against at least one sample from a different excretion study. Retention times, along with product ion ratios, were evaluated according to the WADA TD2021IDCR requirements, in order to determine maximum detection and identification time windows for each metabolite. Established identification windows obtained after LC-MS/(MS) analysis were further compared with those obtained after GC-MS/(MS) analysis of the same samples from the same excretion studies, for the most common analytes monitored by GC-MS/(MS). Full validation was performed for the developed derivatization and shoot method for the identification of methenolone metabolite, 3α-hydroxy-1-methylen-5α-androstan-17-one-3-glucuronide (mth3). Overall, the GRT derivatization presented herein offers a tool for the simultaneous sensitive detection of free, intact glucuronide and sulfate metabolites by LC-MS/(MS) that enhance significantly the detection and identification time windows of specific methenolone and mesterolone metabolites for doping control analysis.

Effect of an anabolic steroid (Metenolon) on contractile performance of the chronically stimulated latissimus dorsi in sheep.

In 12 sheep the left latissimus dorsi muscles (LD) were conditioned by chronic electrostimulation with a pulse generator (Itrel, Medtronic). Six animals (group B) received a weekly intramuscular injection of an anabolic steroid (Metenolon). After 14 weeks the contraction parameters of the left LDs (group A and B) and right LDs (control group) were investigated. The increase in weight of the conditioned LDs was 11.07% (+/- 1.06%) in group A and 79.97% (+/- 40.8; P < 0.05) in group B. The force capacity under stimulation patterns which were just tetanic was 1.15 kp in group A and 4.13 kp in group B (P < 0.05); under supramaximal stimulation patterns it was 4.23 kp (A) and 6.0 kp (B) (P = ns). The force time relation (dF/dt) was 6.7 kp/s for the left LDs in group A versus 16.4 kp/s for the right LDs (P < 0.01); in group B it was 5.13 kp/s for the left LDs versus 15.8 kp/s for the control muscles (P < 0.05). The maximal force (Fmax) per 100 g muscle weight did not differ significantly (A: 2.42 kp/100 g; B: 2.52 kp/100 g). In conclusion, the LD muscles which were subjected to both anabolic therapy and electrical stimulation showed a significant increase in their force capacity due to an enormous increase in mass. Fibre type transformation was complete only in group B. No fibre deterioration was observable in either group. No anabolic side effects were detected in the animals. With the use of anabolic steroids, therefore, a clearer direct increase in contractility on the left ventricle should be expected ("squeezing" theory), as well as a contribution to reduction in wall tension and myocardial oxygen consumption, respectively, according to Laplace's Law (via the considerable increase in thickness).

Effects of treatments by calcium and sex hormones on vertebral fracturing in osteoporosis.

Lateral radiographs of the thoracic and lumbar spine were taken periodically in 49 patients with osteoporosis. Thirty patients were postmenopausal, and 19 nonmenopausal with osteoporosis due to steroids, male hypogonadism, alcoholism, thyrotoxicosis or unknown cause. Patients were studied before, during and after treatment with high calcium alone, or with combined calcium and sex steroids. Calcium was given as effervescent calcium lactate gluconate, and sex hormones as oestradiol valerate, testosterone oenanthate, or methenolone oenanthate. A total of 964 films covering 409 patient-years were available for measurement. On each vertebra, deformity due to loss of anterior height was measured and assigned to one of four grades. For the time interval between each consecutive pair of films, a patient's vertebral fracture rate score was calculated and expressed per thousand patient-years. In comparison with the corresponding pretreatment fracture rate score, both the postmenopausal and the nonmenopausal groups who had not received sex hormones previously, failed to show significant changes (p = 0.144; p = 0.017) on high calcium alone during mean periods of 4.3 and 2.8 years respectively. If the first 2 years on high calcium were excluded for the postmenopausal group, they still failed to show a reduction in fracture rate score (observed for a mean period of 5.0 years; p = 0.04). When treated with combined calcium and sex hormones, both postmenopausal and nonmenopausal groups showed a lower fracture rate score of 20 and 207 respectively when compared with the pretreatment levels of 1500 and 1697 (in mean treatment periods of 3.2 and 4.4 years; p less than 0.001 in each case). When given high-dose calcium alone, but after treatment with sex hormones as well, the postmenopausal group showed no change in fracture rate score from pretreatment (in a mean of 3.1 years; p = 0.069); however the nonmenopausal group still showed a significant reduction in fracture rate score from 1697 to 42 over a mean period of 2.3 years (p = 0.001). The postmenopausal group, after stopping all treatment, showed a higher fracture rate score of 1286 (in a mean of 2.6 years) than did those on combined calcium and sex hormones, in whom the fracture rate score was 20 (in a mean of 3.2 years; p = 0.008). A subgroup of 11 patients with osteoporosis of both the menopausal and nonmenopausal types, had data both before (in a mean of 5.5 years) and during (for a mean of 2.5 years) treatment with calcium alone; the fracture rate scores were 1473 and 918 (p = 0.247).(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of testosterone propionate and methenolone enanthate on the healing of humeral osteotomies in the Wistar rat.

A randomized blind prospective study was carried out to determine if an anabolic androgenic steroid with a high anabolic/androgenic ratio, Group A, (1/0.05) methenolone enanthate (me), compared to an anabolic/androgenic agent with a low anabolic/androgenic ratio, Group B, (1.0/1.0) testosterone propionate (tp), compared to a control, Group C, cottonseed oil (co), affected midhumeral osteotomy healing in 100 two-month-old female Wistar rats. The rats received 4 mg/kg me, 4 mg/kg te, and equal volumes of co weekly. The rats were sacrificed at 2, 4, and 6 weeks. The entire humerus with the healing osteotomy was carefully dissected until all soft tissue attachments were stripped. The healing callus was then subjected to (1) biochemical analysis (hexosamine, hydroxyproline, and calcium), (2) biomechanical testing (progressive distraction of the callus at 1 mm/min on an electrohydraulic materials test system, model 1331, Instron Corp, Canton, MA, and (3) histology. Results of the biochemical testing demonstrated that the percentage of calcium in the healing callus at 2 weeks in group B (tp) was 7.3 +/- 1.0, and this value was greater than that in group C (co), 4.8 +/- 1.6 (p greater than .01), and greater than that in group A (me), 5.6 +/- 0.6 (p greater than .01). At 4 weeks, the percentage of calcium in the callus in group B (tp) was 6.8 +/- 1.9, in group A (me) 7.3 +/- 3.7, and these values were both greater than that in group C (co), 3.9 +/- 2.2 (p greater than .02 and .01, respectively). At 6 weeks the percentage of calcium in the callus in group B (tp) was 11.7 +/- 3.9 and in group A (me) 12.7 +/- 3.9, and again these values were both greater than that in group C (co), 6.7 +/- 2.6 (p greater than .02 and .01, respectively). The remainder of the biochemical analysis, hexosamine and hydroxyproline content, did not show a statistical difference in groups A, B, and C at 2, 4, and 6 weeks. The biomechanical studies and histology also failed to show statistical differences between the three groups at 2, 4, and 6 weeks. The conclusion of this study is that an agent with a low androgenic activity does not increase calcium callus concentrations early in the course of fracture healing compared to an agent with higher androgenic activity. As healing progresses, both agents increase the concentration of calcium in osteotomy healing. The clinical significance of this study is that agents with low androgenic activities favorably influence osteotomy healing and may be clinically useful because they lack unwanted virilizing activity.